PROJECT SUMMARY Aberrant epigenetic regulation is a major contributing mechanism to human disease. Variable DNA methylation of a locus encoding a polymerase III transcribed, 101 base pair non-coding RNA, nc886, is associated with survival of AML patients, body-mass index, Parkinson's disease, and a variety of cancers. Multiple independent studies have demonstrated that the nc886 locus is maternally imprinted, and there is natural variation of this imprinting. Seventy-five percent of individuals have monoallelic DNA methylation, or imprinting, of nc886 and 25% lacked DNA methylation on both alleles. Previous work focused on DNA methylation at the nc886 promoter. However, the region of polymorphic imprinting extends to 1.7 kb flanking nc886 to include a potential enhancer element and a variably methylated CTCF binding site on the centromeric side (cent-CTCF). This larger region of polymorphic imprinting was defined as the nc886 differentially methylated region (DMR). Intriguingly, a C/A SNP lies in a CpG site in the binding motif of the cent-CTCF and influences local DNA methylation and CTCF binding. The central hypothesis is that a C/A SNP influences the likelihood that the nc886 DMR will be imprinted, therefore alerting local chromatin structure and gene expression patterns. Specific Aim 1 is to characterize how DNA methylation of the nc886 DMR alters genome-wide gene expression. While previous studies have modulated nc886 expression and examined changes in genome-wide gene expression, results are contradictory. Additionally, the nc886-associated enhancer and cent-CTCF site have not been investigated. To determine the consequences of DNA methylation at the nc886 DMR, the following elements will be systematically removed (using CRISPR/Cas9) in cell lines with known DNA methylation status: (1) cent-CTCF binding site (2) nc886 coding sequence (3) nc886-associated enhancer, and (4) entire DMR. Consequences of disrupting elements of the nc886 locus will be determined by RNA- sequencing. Specific Aim 2 is to define how variable DNA methylation affects CTCF binding and chromatin architecture. Using cell lines with variable DNA methylation and expression of nc886, Chromatin Immunoprecipitation and Nucleosome Occupancy and Methylome Sequencing (NOMe-Seq) will be used to determine how DNA methylation and SNP status alters CTCF occupancy. HYbrid Capture Sequencing (Hi-C2) will be utilized to determine chromatin looping to our region of interest in cells lines that are biallelically methylated, monoallelically methylated, and biallelically unmethylated at the nc886 DMR. This systematic approach to elucidating the relationship between a common SNP, DNA methylation, chromatin architecture, and gene expression will provide understanding for the potential function of the nc886 DMR. Considering that variable DNA methylation of nc886 is associated with several human diseases, our findings could provide valuable information for the development of obesity, cancer, Parkinson's disease and potentially other yet identified maladies.